The invention relates to a method of generating fragment ions in collision processes and storing light fragment ions in RF ion traps, in which usually high RF storage voltages are set and fragment ions below a relatively high cut-off mass cannot be stored.
3D Paul traps comprise a hyperbolic ring electrode and two rotationally symmetric hyperbolic end cap electrodes. If an electric voltage is applied to the end caps, on the one hand, and to the ring electrode, on the other, an essentially quadrupolar field is generated in the interior. If the voltage is an RF voltage, the RF electric field created is able to store ions. For practical reasons, only one phase of this RF storage voltage is usually applied to the ring electrode, while the end cap electrodes are kept at ground potential. The RF storage voltage usually has a frequency of around one megahertz.
According to Hans Dehmelt, the RF storage field of a 3D ion trap can be envisaged as a three-dimensional pseudopotential well, with a potential minimum in the center, and the potential increasing parabolically in all spatial directions. The ions in the potential well are able to orbit in ellipses or oscillate through the center. The pseudopotential is a temporal integration over the square of the field intensity; the gradient of the pseudopotential continually drives the ions back to the center of the ion trap, irrespective of the polarity of their charge.
The ions are only stored if their mass is above a cut-off mass, however. The term “mass” here is always to be understood as the mass-to-charge ratio m/z, as is required in mass spectrometry, i.e. the physical mass m divided by the number z of (positive or negative) elementary charges. Ions below the cut-off mass are so light that during a half-phase of the RF storage voltage they can already be accelerated up to the opposing electrodes; it is not possible to store them because the pseudopotential does not exist in this case.
The remaining ions oscillate in the pseudopotential well in the ion trap, the oscillation frequencies being roughly inversely proportional to the mass of the ions at a given RF voltage. The oscillation frequencies are one characteristic of the mass. The oscillations of the ions can be resonantly excited very precisely and mass-selectively, for example. Very good approximation formulae are available for the relationship between oscillation frequency and mass.
If the ion trap is filled with a collision gas at a pressure between 1 and 10−2 Pascal, the oscillations of the ions in the potential well are damped within a short time in such a way that the ions collect in a small cloud in the minimum of the potential well. The size of the cloud is determined by the Coulomb repulsion between the ions themselves, on the one hand, and by the centrally-directed force of the pseudopotential, on the other. The time required for the damping is inversely proportional to the pressure of the collision gas. At a pressure of around 10−2 Pascal, the time up to the damping is a few milliseconds; the ion undergoes a few hundred collisions in this time.
3D ion traps can also be used as mass spectrometers by ejecting the stored ions selectively according to their mass and measuring them with secondary electron multipliers. Several different methods of ion ejection have been reported; they will not be discussed here further. Good commercial ion trap mass spectrometers have a mass range of up to a mass-to-charge ratio of m/z=3000 Daltons, and special scanning methods can also resolve the isotope patterns of quadruply charged ions at any mass. Ion trap mass spectrometers are among the most reasonably priced mass spectrometers; they are very widespread.
2D ion traps comprise rod-shaped electrode pairs arranged in parallel; they also go back to Wolfgang Paul. The phases of an RF voltage are applied across each of the electrode pairs. These ion traps are called two-dimensional because the pseudopotential increases from the longitudinal axis in only two spatial directions, and drives the ions back to the axis. In the longitudinal direction, special measures must be employed in order to keep the ions inside the 2D ion trap; these measures can be DC electric fields, or pseudopotentials produced by RF voltages across suitably shaped electrodes. If the ion trap is charged with damping gas, the ions can again be collected; in this case they collect in an elongated ion cloud in the axis of the ion trap.
A special form of 2D ion trap is the quadrupole ion trap, comprising four pole rods. These special 2D ion traps are often called “linear ion traps”. Linear ion traps with four pole rods form a quadrupole field in the interior and can be used as mass analyzers in the same way as 3D ion traps. Here too, there are different scanning methods, for example those which eject the ions mass-selectively through slits in the pole rods or through diaphragms at the end of the rod systems. Commercial instruments with ejection through slits in the pole rods currently have a mass-to-charge ratio range of up to m/z=2000 Daltons.
All ion traps can also be coupled with other types of mass analyzer in order, for example, to achieve particularly high mass resolving power or to be able to measure ions of particularly high mass-to-charge ratios m/z. Couplings with ion cyclotron resonance analyzers, time-of-flight mass analyzers, magnetic sector field devices and Kingdon cells have been described. In these cases, the RF ion traps usually serve only to be available for the fragmentation of the ions, including the collision-induced decompositions which are particularly considered here.
We now turn to a field of application in which mass spectrometry plays an important role: proteomics. This frequently involves enzymatically breaking down the proteins to digest peptides, and analyzing these mass-spectrometrically. Fragmentation processes are very important here because they allow sequences of amino acids and modification structures to be identified.
Nowadays, two fundamentally different types of fragmentation are available in the different types of ion trap: “ergodic” fragmentation and “electron-induced” fragmentation.
All “electron induced” fragmentation methods consist in neutralizing an associated proton of a multiply charged peptide or protein ion by capture or transfer of an electron, whereupon a spontaneous rearrangement leads to cleavage of the amino acid chain. The cleavage does not occur at the peptide bonds, but at neighboring bonds, leading to so-called c- and z-fragment ions. Various forms of this type of fragmentation are now known: electron capture dissociation (ECD); electron transfer dissociation (ETD) and electron transfer by highly excited neutral particles (MAID=“metastable atom induced dissociation”). Electron transfer dissociation (ETD), in particular, is carried out in ion traps.
“Ergodic” fragmentation of analyte ions refers here to a process in which a sufficiently large excess of internal energy in the analyte ions leads to fragmentation. This excess of energy can, for instance, be generated by a large number of collisions between the analyte ions and a collision gas, or by the absorption of a large number of photons from infrared radiation (IRMPD=“infrared multi photon dissociation”), for example.
According to the “ergodic hypothesis” originally formulated by Boltzmann, in a closed system such as that of a complex molecular analyte ion, when a certain energy is present, then every state that can be achieved with this energy will in fact be achieved over the course of time. This ergodic hypothesis has since been proved mathematically, and is therefore no longer strictly a hypothesis. Since the fragmentation represents a possible state, that is the generation of two smaller particles of the analyte ion, the fragmentation will occur at some stage. The absorption of energy temporarily creates “metastable” analyte ions, which then at some stage decompose. The decomposition itself is characterized by a “half life”, although this depends on the quantity of excess energy and cannot be determined with certainty.
The probability that a given bond will experience an ergodic cleavage depends on the binding energy. Only the weakest bonds in the analyte ion will be cleaved with a high probability. In proteins, the weakest bonds are those known as “peptide bonds” between the amino acids, leading to fragments in the b and y series, which occur partly as charged fragment ions and partly as neutral particles. Since the peptide bonds between different amino acids have somewhat different binding energies, some peptide bonds in the analyte ion are cleaved with a greater probability and others with a lower probability. As a result, not all the fragment ions created by cleaving peptide bonds have the same intensity in the fragment ion spectrum. The fluctuations in the intensities thus reflect the energies of the different types of peptide bond. Non-peptide bonds, on the other hand, are cleaved so rarely that the resulting fragments are not found in measurable quantities.
To generate fragment ions from the analyte ions by collision-induced decomposition in RF ion traps, it is necessary to first select an ion species to be fragmented into fragment ions and then measured. The analyte ions are usually present as a mixture: they may originate from several substances, all of which must be analyzed, or they may consist of ions of several charge levels, one of which is to be selected for the fragmentation. The fragment ions (of the first generation of fragmentations) are frequently termed “daughter ions”, and the ions of the ion species selected from the analyte ions for fragmentation are frequently termed “parent ions”. After selecting the parent ions, all other ions located in the ion trap are ejected from it using known methods so that only the parent ions remain.
Incidentally, the parent ions do not all have to have precisely the same mass; it is also possible to use ions of different mass which have the same molecular formula for their elemental composition but include different isotopic combinations. Indeed, the joint fragmentation of all the ions of such an isotope group is the predominantly used method because then the daughter ions also occur in isotope groups, and it easy to read off the charge state on the isotope groups.
The process of ejecting all ions not selected is frequently termed “isolation” of the parent ions. The basic principles of the ejection are largely known, and it can easily be conducted in all commercially available ion trap mass spectrometers. It is based, on the one hand, on using the lower mass limit to eject the ions which are lighter than the parent ions, and, on the other hand, on using mass-selective resonant excitation of the oscillations of the undesired heavier ions; the excitation used is so strong that the ions touch the electrodes and are thus discharged, or otherwise disappear from the ion trap. In 3D ion traps, the resonant excitation is brought about by an alternating voltage, applied across the two end cap electrodes and thus generates a dipole alternating field. In 2D ion traps, the dipolar excitation voltages can be applied across two opposing pole rods.
The remaining parent ions are damped in the collision gas and thus collect again in a small cloud in the center of the ion trap. They can now be fragmented. The usual type of fragmentation is collision-induced decomposition (CID). A relatively soft resonant excitation forces them to oscillate, leading to a large number of low-energy collisions with the collision gas. In many of these collisions, small portions of energy are transferred into the parent ions. The internal energy of the internal molecular oscillation systems increases until one of the weaker bonds within the molecular structure of the parent ion breaks open. A singly charged parent ion forms a daughter ion and a neutral particle; a doubly charged parent ion frequently (but not always) forms two singly charged daughter ions. Since the daughter ions are no longer resonantly excited because they have a different mass, and hence a different oscillation frequency, their oscillations are damped by the collision gas after the fragmentation, and the daughter ions collect in the center. They can then, for example, be measured as a daughter ion spectrum in the conventional way in a detector located outside the ion trap after being resonantly and selectively ejected in sequence according to their mass.
This type of collision-induced decomposition has disadvantages, the main one being that there are both heating and cooling collisions. The terms “heating” and “cooling” refer to the internal energy of the parent ions, not to the energy of the secular oscillations, for which the terms “excite” and “damp” are always used here. If the collisions are all low-energy, i.e. if parent ion and collision gas molecule collide very slowly with each other, the cooling collisions predominate: the internal energy of the parent ions is reduced by transferring energy to the collision gas molecule. The parent ion can only absorb energy in hard collisions of rapidly colliding partners. Since helium is usually used as the collision gas, the collision gas molecules cannot take up any internal energy at all. This transfer of energy into the interior of the parent ions requires the excitation of energy states of the internal oscillation systems, which, as is known from quantum theory, demands a minimum energy. Only hard collisions, i.e. collisions in which the collision partners have a high relative speed, result in the parent ions being heated.
The excitation for the fragmentation must therefore involve collisions which are sufficiently hard, i.e. minimum speeds of the parent ions produced by excitation of their secular oscillations. These hard collisions can only be achieved with a relatively high RF storage voltage because only then are the walls of the potential well steep and high enough to achieve rapid, wide oscillations. But, even in this case, special care must be exercised: the damping of the secular oscillations of the parent ions in the collision gas must always remain in equilibrium with the constant increase in amplitude caused by the dipolar excitation because, if it does not, the amplitudes of the secular oscillations increase until the parent ion leaves the ion trap. Since the damping brought about by collisions with the collision gas is a statistical process, the alternating voltage used to resonantly excite the secular oscillations must be carefully kept small to avoid losing large numbers of ions. Despite reduced average deflection, a high RF storage voltage has to be applied to achieve high collision speeds.
There is rule of thumb in the literature which states that the fragmentation can only be carried out when the RF storage voltage has at least a value which produces a lower mass threshold equivalent to a third of the mass-to-charge ratio of the parent ions. However, this does not allow small fragment ions with a mass-to-charge ratio below a third of the mass-to-charge ratio of the parent ions to be collected in the trap.
Again we turn to proteomics. As already mentioned, this field frequently involves enzymatically breaking down the proteins to digest peptides, and analyzing these mass-spectrometrically. If one begins with peptide ions, then often so-called internal fragments form in the collision cells; these internal fragments originate from two cleavages of the amino acid chain. So-called immonium ions, in particular, often result; these are charged single amino acids originating from somewhere in the chain. The measurement of such immonium ions has high informational value since they immediately signalize the presence of this amino acid in the peptide. It is frequently possible to read off the amino acid composition of the peptide from the immonium ions, even if it is not possible to thus determine the arrangement of the amino acids along the chain.
To also store very small fragment ions (particularly of the immonium ions) by collision-induced decomposition, special methods have recently been published which utilize the slow, metastable decomposition of the ions through the ergodic fragmentation process.
In U.S. Pat. No. 6,949,743 B1 (J. C. Schwartz) a method of collision-induced decomposition is proposed which uses a brief excitation of the analyte ions with a short pulse of resonant alternating excitation voltage at high RF storage voltage. The RF storage voltage is then decreased in order to lower the lower mass threshold and to allow light fragment ions to be collected.
In Patent DE 10 2005 025 497 B4 (A. Brekenfeld), too, the fragmentation is carried out for a brief period of between a few tenths of a millisecond and a few milliseconds at an RF storage voltage which is higher than normal. In the course of the subsequent damping of the secular oscillations by the collision gas, there is then a controlled changeover to a low RF storage voltage. This transition to low RF storage voltages must not occur rapidly because, if it does, the oscillating ions can escape from the well as the storage well becomes shallower. When the high RF storage voltage for the fragmentation is applied, it is possible to use either resonant excitation or deflection of the parent ions far out of the center by means of DC potentials across at least one of the electrodes; when these are switched off only the strongly restoring force of the RF storage voltage continues to act on the ions, so that the ions undergo powerful collisions with the collision gas. The subsequent low RF storage voltage allows the fragment ions, which also include very light daughter ions and granddaughter ions, to then collect in the center of the ion trap, and they can be measured in the normal way.
Heavy analyte ions in the mass range of m=2000 to 5000 Daltons and above represent a great difficulty for collision-induced decomposition. This is especially because they require much more internal energy to fragment ergodically in a reasonably short time. If there is a longer delay before fragmentation, the collision gas again causes cooling, i.e. there is a loss of internal energy so that no further ergodic fragmentation occurs at all. In addition, a great many highly charged fragment ions are produced, which make it difficult or even impossible to evaluate the fragment ion spectrum. One solution to this problem is to deprotonate the highly charged analyte ions before they are fragmented.
An interesting method of deprotonating highly charged pseudomolecules, which has basically been known for a long time, has recently been elucidated. The highly charged pseudomolecule ions of a substance, which are present with different charge levels, can be deprotonated at the same time in an RF ion trap, and this deprotonation process can be halted at a certain charge level so that all pseudomolecule ions with higher charge levels collect at this selected charge level in a partially deprotonated state. This requires that a gentle resonant excitation of the secular oscillations be created at the mass-to-charge ratio m/z of this charge level of the analyte ions by means of a dipole alternating voltage. The ions of this charge level, which are now oscillating in an excited state, are no longer able to take part in further reactions with deprotonating reactant anions, since deprotonation requires the partners involved to have a low relative speed. This method is described in U.S. Pat. No. 7,064,317 B2 (S. M. McLucky et al.).
Such a conversion of highly charged pseudomolecular ions of different charge levels into a predetermined charge level provides, at the same time, a high sensitivity because the analyte ions of all higher charge levels collect with a relatively high yield at the selected charge level during deprotonation. Yields of over 50 percent can be achieved. If the highly charged ions of several substances are present, it is thus also possible to select the analyte ions because the ions of the foreign substances are not collected, but are deprotonated to the bitter end, until they are neutralized, if the reaction time is long enough.
This type of partial deprotonation is therefore very useful in making highly charged heavy analyte ions available for a collision-induced decomposition in the first place.